Fusobacterium nucleatum outer membrane proteins Fap2 and RadD induce cell death in human lymphocytes

Abstract

Bacterially induced cell death in human lymphocytes is an important virulence factor for pathogenic bacteria. Previously discovered mechanisms of bacterially induced cell death are predominantly based on the transfer of bacterial proteins to the target host cell, such as the toxins secreted through type I, II, and VI secretion systems or effector proteins injected through type III, IV, and Vb secretion systems. Here, we report a mechanism employed by the Gram-negative oral pathogen Fusobacterium nucleatum for cell death induction of human lymphocytes via two outer membrane proteins (OMPs), Fap2 and RadD, which share regions homologous to autotransporter secretion systems (type Va secretion systems). Genetic and physiological studies established that inactivation of the two OMPs led to significantly reduced ability to trigger cell death in Jurkat cells, while the corresponding double mutant was almost completely attenuated. Additional biochemical and molecular analyses demonstrated that cell-free F. nucleatum membranes are sufficient to induce cell death in Jurkat cells, suggesting that no active process or effector protein transfer was necessary to induce eukaryotic cell death.

FIG. 1.

Transcriptional analysis of fap2 from F. nucleatum PK1594. Relative cDNA abundance of fap2 was determined for cells grown in broth culture to logarithmic or stationary phase, on agar plates, or in the presence of Jurkat cells (4-h incubation) to logarithmic phase. n = 3. ND, not determined; *, P ≤ 0.01 compared to logarithmic phase results.

FIG. 2.

Jurkat cell death induction by F. nucleatum. Cell death rates were quantified based on the percentage of annexin V-FITC-positive Jurkat cells in samples incubated with F. nucleatum whole cells or membranes. F. nucleatum ATCC 23726 cells and membranes induced cell death in ∼75% and 41% of Jurkat cells, respectively, in the annexin V assay, and these numbers serve as the basis for the relative calculations presented. n ≥ 3. P ≤ 0.01 for all conditions compared to the wild-type control (wt).

FIG. 3.

Membrane preparation of F. nucleatum strains. Samples were boiled for 10 min in SDS gel loading buffer and resolved by 4% SDS-PAGE and Sypro Ruby staining. An illustration of the protein bands present in the wild-type strain is indicated on the right. Protein bands missing in mutant strains are indicated with arrowheads and labeled on the protein band illustration to the right. Molecular mass standards (marker) are indicated.

FIG. 4.

Transcription of fap2 and its downstream ORF, Fn1448, in wild-type F. nucleatum and the mutant derivative lacking fap2. F. nucleatum cells were grown to mid-exponential-growth phase to ensure fap2 expression. fap2, but not Fn1448, expression in the mutant was significantly different (P ≤ 0.01) from that in the wild type. n = 3.